The use of .alpha.-amylases for enzymatic conversion of starch into sugars, e.g. for the production of fuel alcohol or High Fructose Syrup (HFS), is widely practised. Among industrial starch liquefaction processes jet-cooking is the almost globally preferred mode of starch liquefaction, but for the performance of this process thermostable .alpha.-amylases are required.
HFS is manufactured from high DX syrups, the term DX meaning percentage by weight of dextrose (D-glucose) calculated on the basis of dry substance (DS) of syrup. The overall enzymatic liquefaction process generally adopted for conversion of starch into high DX syrup is a two-stage process. The first step is the liquefaction, i.e. the hydrolysis of starch into a mixture of oligosaccharides, the so-called maltodextrins. This process is catalyzed by .alpha.-amylases, and in a typical jet-cooking process the starch slurry is heated for at least several minutes to 105.degree.-110.degree. C., usually with a single dose of .alpha.-amylase, and then held at about 90.degree. C. for at least 1 hour. In the primary stage of the overall liquefaction, gelatinization and mechanical thinning of the starch slurry are effected. Further degradation (dextrinization) occurs in the secondary stage of the process. With respect to the jet-cooking process, reference is made to U.S. Pat. No. 3,912,590.
Hitherto mostly preferred thermostable .alpha.-amylases for industrial liquefaction processes are of bacterial origin and from the genus Bacillus. Thus a well adapted .alpha.-amylase for use in the jet-cooking Process is TERMAMYL.RTM. from Bacillus licheniformis, supplied by NOVO NORDISK A/S, Denmark. .alpha.-amylases from Bacillus stearothermophilus are disclosed in U.S. Pat. Nos. 2,695,683 and 4,284,722. A Bacillus stearothermophilus .alpha.-amylase (THERMOLASE.TM.) is available from Enzyme Development Corporation, NY, USA.
Due to the Properties of the .alpha.-amylases hitherto available, the liquefaction process is typically performed at pH about 6.0-6.5. At pH below 6 the amylolytic activity rapidly decreases, and at pH above 6.5 the formation of unwanted byproducts such as maltulose or maltulose "precursors" become troublesome. Bacillus licheniformis .alpha.-amylase, for instance, is rapidly inactivated at pH values below 6.0. Moreover it requires at least 50 ppm calcium for stabilisation when used for industrial starch liquefaction, and it is completely inactivated at 120.degree. C. The Bacillus stearothermophilus .alpha.-amylase has certain advantages over the Bacillus lichenformis enzyme, notably a lower pH optimum. However these enzymes are not fit for starch liquefaction at pH values below 5.
The subsequent saccharification step, in which the maltodextrins are converted into dextrose, is mostly catalyzed by a glucoamylase enzyme. Commercial glucoamylase preparations, usually derived from Aspergillus or Rhizopus species, are available from various manufacturers, e.g. as AMG.TM. 200 L, a product obtained from Aspergillus niger and manufactured by NOVO NORDISK A/S, Denmark. These glucoamylase enzymes operate optimally at pH 4.0-4.5.
Now hyperthermophilic archaebacteria have been isolated from solfataric and submarine hydrothermal systems (Fiala, G. & Stetter, K. O.; Arch. Microbiol., 145, 56-61 (1986) and Kelly, R. M. & Deming, J. W., Biotech. Progress, 4, 47-62 (1988). The most extreme thermophilic bacteria known so far belong to the genera Pyrococcus, Pyrodictium and Pyrobaculum. It has been presumed that members of Pyrococcus contain heat stable proteases and amylases (Stetter, K. O., J. Chem. Technol. Biotechnol., 42(4); 315-317 (1988)). Growth conditions for Pyrococcus woesei have been examined (Zillig et al.; Syst. Appl. Microbiol., 9, 62-70 (1987)). However, amylases from Pyrococcus have never been isolated or investigated.
In order to eliminate the need for intermediate pH adjustment the art has long sought for thermostable starch liquefaction enzymes capable of liquefying at pH as low as 4.0. Moreover it is preferred to avoid addition of calcium salts in order to reduce purification costs. Therefore it is an object of the present invention to obviate the shortcomings of the .alpha.-amylases known heretofore by furnishing a novel hyperthermostable .alpha.-amylase that has a low pH optimum, and that is essentially independent of calcium ions.